With as many as 1030 microbial genomes globally, across multiple different environmental and host conditions, genetic variety both within and between microbiomes is well recognized (Huse et al., PLoS Genetics 4(11): e1000255 (2008)). As a result of this variety, characterizing the contents of a microbiome is a challenge for current approaches. First, standard culturing techniques are successful in maintaining only a small fraction of the microorganisms in nature. Means of more direct profiling, such as sequencing, face additional challenges. Both the sheer number of different genomes in a given sample and the degree of homology between members presents a complex problem for identification of species.
Prokaryotic ribosomes are composed of two subunits: a 30S subunit (small subunit) and a 50S subunit (large subunit), which together make up the complete 70S ribosome, where S stands for Svedberg unit for sedimentation rate. The 30S subunit is composed of 16S ribosomal RNA and 21 proteins, while the 50S subunit is composed of two rRNA species, the 5S and 23S rRNAs. The presence of hyper variable regions in the 16S rRNA gene provides a species specific signature sequence which is useful for bacterial identification process. 16S ribosomal DNA sequencing is widely used in microbiology studies to identify the diversity in prokaryotic organisms as well as other organisms and thereby study the phylogenetic relationships between them.
16S RNA genes from microbiota have been used to characterize microbial communities from diverse environments such as soil and gastrointestinal samples. However, methods to improve detection of microorganisms, including increasing the ability to detect microorganisms present in small numbers, identifying and distinguishing microbiota with both high and low conservation of 16S sequences, and improving accuracy of reads, are desired.